Apparatus and method for sizing nanoparticles based on optical forces and interferometric field detection

ABSTRACT

Light from a laser source is split into a reference arm and a detection arm. The light in the detection arm is focused into a channel containing particles to be detected and is backscattered by the particles. The light in the reference arm is attenuated. The attenuated and backscattered light are caused to interfere and detected by a split detector so that the effects of background light can be subtracted out, while the backscattered light is detected to detect the particles.

REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional PatentApplication No. 60/677,411, filed May 4, 2005, whose disclosure ishereby incorporated by reference in its entirety into the presentdisclosure.

STATEMENT OF GOVERNMENT INTEREST

The work leading to the present invention was supported by NSF Grant No.PHS-0441964 and DARPA Grant No. MDA972-00-1-0021. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed to a technique for the detection ofnanoparticles, such as viruses, and more particularly to an opticaltechnique using interferometry which lessens the dependence on particleradius relative to known techniques.

DESCRIPTION OF RELATED ART

Particles with characteristic sizes of less than 100 nm are becomingincreasingly important in the context of nanoscience and technology.Applications range from solid-state physics to biology. For example,semiconductor nanoparticles are used as single photon emitters inquantum information science and as fluorescent markers for biologicalprocesses. Similarly, noble metal particles are used as contrast agentsin microscopy, as biochemical sensors, as probes in scanning probemicroscopy, or as nonbleachable biological labels. Furthermore,specially engineered particles such as nanoshells are employed forphoto-thermal tumor ablation and for cancer therapies. Polymernanoparticles are being used as calibration standards and, infunctionalized form, also as probes in biological imaging. There arealso various naturally occurring nanoparticles of high societal impact.Among them are carbon particles originating from combustion or differentsorts of infectious viruses.

Because of their small size, nanoparticles are not easy to detect, andit is evident that there is high demand in novel techniques for thereliable detection, characterization, sorting, and tracking of nanoscaleparticles of various sorts. In public health, for example, there isconcern about the impact caused by the accelerating rate of nanoparticleemissions and waste. It has been determined that the inhalation ofultrafine particles originating from emissions of various kinds cancause heritable mutations. The development of nanoparticle sensors isalso a high priority for environmental monitoring and for the detectionof various agents used in bioterrorism. Furthermore, as the feature sizeof integrated circuits becomes increasingly smaller, contaminationcontrol of ultrafine particles poses a challenge for the semiconductorindustry.

Among the different detection strategies, optical techniques areespecially attractive because of their noninvasive nature,high-sensitivity, and potential for realtime detection. Most of theoptical schemes rely on the detection of scattered light from anensemble of particles. However, the detection of single nanoparticles isa challenging task which, so far, has been only accomplished by indirectmeans, i.e., by fluorescent labeling or immobilization on a surface andsubsequent analysis with dark field microscopy. It has been recognizedthat current real-time single particle detection methods formicrometer-sized particles are not suitable for nanoparticle detectionbecause the intensity of light scattering scales with the sixth power ofparticle size. This rapid decrease of the signal renders small particlesinvisible.

Real-time nanoparticle detection demands an interaction mechanism with aweaker dependence on particle size. One strategy in this directionrelies on detecting the scattered light interferometrically, therebyaccessing the scattered electric field amplitude as opposed to thescattered power. This approach has been demonstrated, almost 20 yearsago, in U.S. Pat. Nos. 5,037,202 and 5,061,070, and recently applied forthe detection of immobilized gold particles as small as 5 nm indiameter. Other detection schemes with an r₀ ³ signal dependence(r₀=particle radius) aim at measuring particle absorption cross sectionsby means of the photothermal effect or measuring optical gradient forcesacting on nanoparticles in strongly focused laser beams.

Although these methods extend the detection sensitivity to smallerparticle sizes, they suffer from other shortcomings which prevent thedetection of single nanoparticles in real time. Either they requireparticle immobilization to ensure sufficiently long acquisition times orthey are subject to a background signal originating from Brownian motionor direct detector exposure.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a technique formeasuring nanoparticles which overcomes the above-noted shortcomings.

To achieve the above and other objects, the present invention isdirected to a background-free detection approach which gives usunsurpassed real-time detection sensitivity for nanoscale particles. Wedemonstrate the successful detection and classification of low-indexparticles such as individual viruses carried in a microfluidic system.In the current version, we are able to detect individualwater-solubilized polymer particles of 10 nm radius within a fewmilliseconds. Our detection scheme is well suited for the screening andsorting of various nanoscale particles such as viruses and largerproteins and is compatible with current microfluidic technology.

The present invention provides a background-free real-time detectionscheme capable of recognizing low-index nanoparticles such as singleviruses in water. The method is based on interferometrically measuringthe electromagnetic field amplitude of the scattered light. A splitdetector is used to generate a background-free signal that rendersunprecedented sensitivity for small particles. In its currentconfiguration the sensor is capable of detecting low-index particles inwater down to 10 nm in radius or single gold particles as small as 5 nm.We demonstrate the detection of such small particles in a microfluidicsystem with a time resolution of 1 ms.

The invention provides a background-free, interferometric detectiontechnique for nanoscale particles. The detector works in real time andwith single particle sensitivity. Interferometric detection ensures thatthe signal amplitude scales with the third power of particle size, andthe use of a split detector ensures the best possible signal-to-noiseratio, independent of laser power noise. Within a one-millisecond timewindow we are able to reliably detect a single 10 nm polystyreneparticle or a single 5 nm gold particle. Even higher sensitivity couldbe achieved by modulating the reference beam length (phase modulation)or by heterodyne detection. The detection scheme will find applicationsin a variety of fields such as particle tracking inside cells, detectionof biowarfare agents (viruses), contamination control of water and air,and others. The detector can also be used as a prescreening stage in alarger biodetector assembly for deciding whether a subsequent one-shotdetector stage with high chemical specificity (antigenantibody,polymerase chain reaction, laser spectroscopy, etc.) should be exposedor not.

BRIEF DESCRIPTION OF THE DRAWINGS

A preferred embodiment of the present invention will be set forth indetail with reference to the drawings, in which:

FIG. 1 shows a schematic rendering of the detector according to thepreferred embodiment;

FIG. 2 shows histograms of signal amplitudes obtained with the detectorof FIG. 1;

FIG. 3 shows an experimental analysis of detection limits; and

FIG. 4 shows dependence of the signal amplitude on particle radius.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A preferred embodiment of the present invention will be set forth indetail with reference to the drawings, in which like reference numeralsrefer to like elements throughout.

The detection scheme is schematically shown in FIG. 1 as 100. Using theelectro-osmotic effect, as shown in FIG. 1, part (b), a particlesolution 102 containing particles 104 is transported through amicrofluidic channel 106. The channel 106 is subdivided by a barrier 108with various nanoscale channels 110. As shown in FIG. 1, part (a), alaser source 112 emits a λ=532 nm laser beam L, which is split by a50/50 beam splitter 114 into two perpendicular paths L₁, L₂. One path L₁serves as a reference for later interferometric recombination in areference arm including an attenuator 116 and a mirror 118, and theother path L₂ is focused with an objective lens 120 (NA=1.4) into asingle preselected nanochannel. In principle, many channels could besampled sequentially or in parallel by making use of a programmablespatial light modulator. The lateral dimensions of the nanochannels arecomparable to the size of the laser focus, ensuring that no more thanone particle crosses the focus at any time. The backscattered light froma particle passing through the laser focus is collected with the sameobjective lens 120 and is then recombined with the reference beam by thebeamsplitter 114 and directed through a lens 122 and a 500 μm pinhole124 onto a split photodetector 126. The power of the reference beam canbe arbitrarily attenuated by the attenuator 116 using a λ/2 plate placedbetween two polarizers or by any other suitable attenuation technique.The signal from the detector 126 is analyzed in a data acquisitionsystem 128 in a manner to be described below.

Plot (c) in FIG. 1 shows a typical detector time trace S(t). Each peakrepresents a single particle passing through the laser focus. Importantelements in the preferred embodiment are (i) interferometric detection,(ii) variable attenuation of the reference beam by the attenuator 116,and (iii) the use of a split detector 126 to ensure a background-freesignal.

To understand the nature of the detector signal, let us denote the fieldof the scattered light as E_(s), and the field of the reference beam asE_(r). When the particle is in the focus, the intensity distribution onthe detector surface is calculated asI=|E _(r)|² +|E _(s)|²+2Re{E _(r) ^(•) E _(s)}.  (1)

The signal S(t) measured by the split detector corresponds to thedifference between two halves of the detector surface normalized by thetotal power incident on the detector, i.e.,${S = \frac{\left( {{\int_{\Subset}{I{\mathbb{d}a}}} - {\int_{\supset}{I{\mathbb{d}a}}}} \right)}{\int_{\circ}{I{\mathbb{d}a}}}},$

with ⊂ and ⊃ denoting the two halves of the photodetector surface and ∘denoting the entire photodetector surface. In the absence of a passingparticle, the reference beam and the light backreflected by opticalelements are adjusted into the center of the split photodetector suchthat the differential signal S(t) is zero. The interference between thereference beam and the backreflected light does not affect our detectionmethod because it is stationary and therefore does not generate anydifferential signal. Thus, S(t) is a background-free signal similar tofluorescence that is commonly used to detect and track single molecules.

When a nanoparticle passes through the nanochannel, the symmetry of thebackscattered light is disturbed, and the detector signal S(t) isdefined by the interferometric term $\begin{matrix}{{S(t)} = {2\quad{Re}{\left\{ \frac{{\int_{\Subset}{E_{r}^{*}E_{s}{\mathbb{d}a}}} - {\int_{\supset}{E_{r}^{*}E_{s}{\mathbb{d}a}}}}{\int_{\circ}{{E_{r}}^{2}{\mathbb{d}a}}} \right\}.}}} & (2)\end{matrix}$

Here, we neglected the scattered light intensity |E_(s)|² in thenumerator which is legitimate as long as the reference field is strongerthan the scattered field. For the same reason, we only retained thereference beam intensity |E_(r)|² in the denominator and rejected allterms in E_(s). These approximations are justified considering the weaksignal scattered by a nanoparticle.

Light scattering from a particle moving through the nanochannel dependson the particle position relative to the center of the laser focusgiving rise to a nonzero signal S(t) recorded by the splitphotodetector. The amplitude of the signal depends on the particle'spolarizability which, in turn, depends on particle size and shape, aswell as on its dielectric properties. As an example, in FIG. 2, plot (a)shows a histogram of signal amplitudes for a mixture of polystyreneparticles of two different sizes, r₀=15 nm and r₀ =40 nm. Thedistribution shown in FIG. 2, plot (b) corresponds to a mixture r₀=7 nmand r₀=20 nm gold nanoparticles. The individual particle distributionsappear clearly resolved, which demonstrates that our detection strategyis well suited for characterization and subsequent separation on aparticle by particle basis. Similar procedures can be applied toseparate biological particles, such as viruses or bacteria. In fact, weare currently able to detect single Influenza A X-31 viruses in realtime and discriminate them from other particles of similar size. FIG. 2,plot (c), shows a histogram of signal amplitudes for a mixture of thatvirus (left peak) and 100 nm polystyrene beads. All data sets have beenacquired in water with each individual detection event lastingapproximately 1 ms.

To quantitatively understand the sensitivity and detection limits, wefirst note that, for a given instant of time, the signal S(t) in Eq. (2)depends linearly on the electric field amplitude E_(s) of the scatteredlight. On the other hand, the scattered field is linearly related to theamplitude of the focused laser field E_(f) and the particlepolarizability α. Hence, the detector signal satisfies the followingproportionality:S(t)∝Re(α)√{square root over (P _(f) /P _(r))},  (3)

where P_(f) and P_(r) are the powers of the focused laser beam and thereference beam, respectively. The proportionality constant depends onthe momentary particle position, on the result of spatial integrations,on various physical constants, and on experimental conditions such asthe numerical aperture of the objective, mirror reflectivity, detectorquantum efficiency, etc. An important fact is that P_(f) and P_(r) areindependent from each other. Thus, the total incident laser power can beincreased and focused to a more intense spot while the reference beamcan be attenuated, thereby increasing the differential signal amplitudeS(t) and allowing even smaller particles to be detected.

FIG. 3, plot (a) demonstrates this property for a sample with r₀=50 nmpolystyrene particles. The plot shows the dependence of the signalamplitude S(t) on the reference beam power P_(r). The red line is a fitaccording to Equation (3). The detector signal increases the more thereference beam is attenuated.

In order to assess the detection limit, we analyze the signal-to-noiseratio (SNR). The noise floor of the detector signal is defined in theabsence of the scattered field. Since the spot of the reference beam ispositioned at the center of the split photodetector, the signal noisedoes not depend on power noise of the laser. Instead, it is defined bythe beam pointing instability and electronic noise of the detector. Thepointing instability causes the beam spot to deviate from its centralposition on the detector, giving rise to a nonzero detector response.Denoting the beam angle with respect to the unperturbed optical axis asθ, the noise level for the differential signal can be expressed asN=√{square root over (P _(v) ²+[θ_(rms) P _(r)]²)}/P _(r),  (4)

where P_(v) represents the “power equivalent” of electronic detectornoise and θ_(rms)P_(r) accounts for the pointing instability of thelaser. When P_(r)>>P_(v), the noise becomes constant and proportional toθ_(rms). However, when P_(r) is attenuated such that θ_(rms)P_(r) isless than or approximately equal to P_(v), the noise level increasesrapidly with decreasing P_(r). Using Equations (3) and (4), we obtain:$\begin{matrix}{{\frac{S}{N} \propto {{Re}\sqrt{\frac{P_{f}P_{r}}{P_{v}^{2} + \left\lbrack {\theta_{rms} + P_{r}} \right\rbrack^{2}}}}},} & (5)\end{matrix}$

which predicts that the best SNR is achieved when the power of thereference beam is P_(r) ^(max)=P_(v)/θ_(rms).

FIG. 3, plot (b) shows the measured average SNR for 50 nm particles fordifferent reference beam powers P_(r). The plot shows the dependence ofthe SNR on the reference beam power. The curve is a fit according to Eq.(5) and demonstrates that the SNR has a maximum as predicted by Eq. (5).The measured detector noise equivalent power is P_(v)=0.7 nW (rms), andthe laser pointing instability is θ=4.5×10⁻⁴ (rms). Those values predicta maximum at P_(r)=1.6 μW, which is in agreement with the fitted curvein plot (b). Several hundred particles were used for each data point. Itturns out that the recipe for achieving the best sensitivity and lowestdetection limit is to increase the laser power while keeping thereference beam at the level of maximum SNR.

The lowest possible reference beam power is determined by thebackscattered light in the absence of a passing particle. Thisbackscattered light is due to the optical index mismatch between thedifferent interfaces and is analogous to background fluorescence insingle molecule experiments. Because this backscattered light interfereswith the scattered light from a passing particle it assumes a similarfunction as the reference beam. When this unwanted backscatteringbecomes stronger than the reference beam power we may simply replaceP_(r) in Eq. (5) by the power of the backscattered light P_(b) andobtain the following limit:Max[S/N]∝Re(α)√{square root over (P _(f) /P _(b))}/θ_(rms)=Re(α)×√{square root over (R)}/θ _(rms),

where, in the last step, we expressed the backscattered light by thefocused beam power P_(f) using a generalized reflectivity R. Thus, thebest possible SNR in our detection scheme is entirely defined by theindex mismatch between the interfaces and the beam pointing instability.Both effects can be minimized in a favorably engineered detector design.

Let us now compare the SNR of our detection scheme with the SNR ofstandard scattering-based detection. According to Eq. (2), the maximumnormalized differential signal amplitude (S=1) is obtained when thephase between E_(s) and E_(r) (or E_(b)) assumes a value whichconcentrates all energy on one half of the split detector. This can onlyhappen if the scattered field amplitude is equal to the amplitude of thereference beam or, equivalently, to the amplitude of the backscatteredbeam, i.e., P_(s)=P_(b). For sufficiently strong powers our SNR becomes{S/N} _(preferred embodiment)=(1/θ_(rms))√{square root over (P _(s) /P_(b))}.

On the other hand, the maximum SNR in standard light scattering can bewritten as{S/N} _(scattering)=(1/η)P _(s) /P _(b),

where η=√{square root over (<dP>²)}/P is the laser power noise. The SNRin our detection scheme is proportional to √{square root over(P_(s)/P_(b))}, versus P_(s)/P_(b) for scattering-based detection, andtherefore proportional to the third power of particle size, versus thesixth power of particle size for scattering-based approaches. Second,the SNR in light scattering depends on laser power noise which cannoteasily be controlled. On the other hand, our scheme depends on theangular pointing stability of the laser which can be controlled, forexample, by reducing the optical path length. Furthermore, thedimensionless pointing stability coefficient θ_(rms) for lasers is muchsmaller (by orders of magnitude) than typical power noise.

In order to verify the r₀ ³ dependence we measured the signal amplitudesof monodisperse particles of different sizes. As shown in FIG. 4, weobtain very good agreement with theory for both polystyrene and goldparticles. In that figure, plot (a) shows the dependence of the signalamplitude on particle radius r₀ for gold particles, while plot (b) showsthe same dependence for polystyrene particles in water. The line in bothplots is a fit according to r_(o) ^(n) with n=2.9±0.3 in plot (a) and2.6±0.3 in plot (b). The threshold for the smallest particle that can bedetected is defined by the choice of the minimum acceptable SNR. Asdemonstrated in FIG. 2, we can reliably detect r₀=15 nm polystyreneparticles and 7.5 nm gold particles in water using a SNR of 3 and adetection bandwidth of B≈10 kHz (one detection event≈1 ms). By choosinga more compact design, better index matching at interfaces, and morestable laser sources we expect to considerably increase the detectionthresholds.

While a preferred embodiment of the invention has been set forth above,those skilled in the art who have reviewed the present disclosure willreadily appreciate that other embodiments can be realized within thescope of the invention. For example, numerical values are illustrativerather than limiting, as are specific techniques for attenuation and thelike. Therefore, the present invention should be construed as limitedonly by the appended claims.

1. A method for detecting a particle in a location, the methodcomprising: (a) emitting a beam of electromagnetic radiation; (b)splitting the beam of electromagnetic radiation into a first componentand a second component; (c) directing the first component into areference arm; (d) directing the second component into the location; (e)receiving light backscattered from the location; (f) causing thebackscattered light to interfere with the first component from thereference arm to produce an interference intensity distribution; (g)detecting the interference intensity distribution with a detectorcomprising a plurality of components; and (h) detecting the particle inaccordance with a difference among detection signals form the pluralityof components.
 2. The method of claim 1, wherein step (h) comprisesderiving a particle detection signal from a difference between thedetection signals from two of said components.
 3. The method of claim 1,wherein the reference arm comprises an attenuator, and wherein step (c)comprises attenuating the first component with the attenuator.
 4. Themethod of claim 3, wherein the attenuator is adjustable, and whereinstep (c) comprises adjustably attenuating the first component with theadjustable attenuator.
 5. The method of claim 4, wherein the adjustableattenuator is adjusted to maximize a signal-to-noise ratio of thedifference among the detection signals as a function of a degree ofattenuation of the first component.
 6. A system for detecting a particlein a location, the system comprising: a source of a beam ofelectromagnetic radiation; a beam splitter for splitting the beam ofelectromagnetic radiation into a first component and a second component;a reference arm receiving the first component from the beam splitter;focusing optics, receiving the second component from the beam splitter,for directing the second component into the location and for receivinglight backscattered from the location, thereby causing the backscatteredlight to interfere with the first component from the reference arm toproduce an interference intensity distribution; a detector comprising aplurality of components for detecting the interference intensitydistribution; and a data acquisition system for detecting the particlein accordance with a difference among detection signals form theplurality of components.
 7. The system of claim 6, wherein the dataacquisition system derives a particle detection signal from a differencebetween the detection signals from two of said components.
 8. The systemof claim 6, wherein the reference arm comprises an attenuator.
 9. Thesystem of claim 8, wherein the attenuator is adjustable.
 10. The systemof claim 9, wherein the adjustable attenuator comprises ahalf-wavelength plate.
 11. The system of claim 6, wherein the focusingoptics comprise an objective lens.